Optical microscope device, microscopic observation method and computer program for microscopic observation using single light-emitting particle detection technique

ABSTRACT

There is provided a microscopic observation technique capable of detecting a light-emitting object or a light-emitting particle moving in a thick sample by the scanning molecule counting method. In the inventive technique, the light from a light detection region of is detected the optical system of a confocal or multiphoton microscope is detected with while moving the light detection region in each observed subregion obtained by dividing a region to be observed into plural regions; the signal of the light from a light-emitting particle is individually detected; and the position of the light-emitting particle corresponding to the detected signal is determined in the region to be observed. The moving of the position of the light detection region in each observed subregion is performed continuously in at least two directions or and/or continuously multiple times in each observed subregion.

TECHNICAL FIELD

This invention relates to optical microscopic observation techniques fordetecting and imaging an atom, a molecule or these aggregate in a liquid(Hereafter, these are called a “particle”), for example, a biomolecule,such as protein, peptide, nucleic acid, lipid, sugar chain, amino acidor these aggregate, a particulate object, such as a virus and a cell; ora non-biological particle, and more specifically, to optical microscopedevices, microscopic observation methods and computer programs formicroscopic observation, using a single light-emitting particledetection technique which detects individually the light from a singleparticle by means of an optical system which can detect light from amicro area in a solution, such as the optical system of a confocalmicroscope or a multiphoton microscope. In this regard, in thisspecification, a particle which emits light (Hereinafter, referred to asa “light-emitting particle”) may be a particle which emits light byitself or a particle to which an arbitrary light-emitting label orlight-emitting probe is attached, and the light emitted from alight-emitting particle may be fluorescence, phosphorescence,chemoluminescence, bioluminescence, scattering light, etc.

BACKGROUND ART

According to the developments of optical measurement techniques inrecent years, there have become possible the observation with an opticalmicroscope and the capturing of a microscopic image (the determinationof the position of a two-dimensional or three-dimensional image) throughthe detection and measurement of faint light at a single fluorescentmolecule level. For instance, as described in patent document 1, etc.,in accordance with a microscope device consisting of a laser scan typeconfocal microscope and a super sensitive photodetector, a condition ina sample is imaged by detecting light from a micro region which is aregion where the light is detected in the microscope (Hereinafter,referred to as a “light detection region”) while moving the lightdetection region in the sample so as to scan the inside of the sample.In the case of the optical system of such a confocal microscope, sincethe light emitted from the outside of the light detection region isblocked and not allowed to reach the photodetector, the detection andmeasurement of the faint light at a single fluorescent molecule level ora single photon level are possible, and therefore, the imaging with suchfaint light becomes possible. Further, as an alternative way of theoptical microscopic observation and the capturing of a microscopic imagewith faint light at a single fluorescent molecule level, as described inpatent document 2, etc., there have been proposed microscope devicesusing evanescent light, also. In the cases of such microscope devicesusing evanescent light, excitation light is given only to a thin regionof about 100 nm near the surface of a cover glass and there occurs nolight emission from regions other than the thin region, and thus,background light in a microscopic image is greatly reduced, and thereby,an image with faint light at a single fluorescent molecule level or asingle photon level will be obtained.

By the way, in patent documents 3-8, etc., Applicant of the presentapplication proposed a new optical analysis technique which enablesdetecting a light-emitting particle distributed or dissolved in liquid(hereinafter, referred to as “scanning molecule counting method”). Inthis scanning molecule counting method, the optical system, such asthose of a confocal microscope and a multiphoton microscope, which candetect light from a micro area in solution, is used, in which theposition of the light detection region is moved, namely, the inside ofthe sample solution is scanned with the light detection region, and whenthe light detection region encompasses a light-emitting particledistributed and moving at random in the sample solution, the lightemitted from the light-emitting particle is detected individually, andthereby, one by one, light-emitting particles in the sample solution aredetected so that the counting of the light-emitting particles and theacquisition of the information on the concentration or the numberdensity of the light-emitting particles in the sample solution becomespossible.

PRIOR TECHNICAL DOCUMENTS Patent Documents

-   [Patent document 1] JP2005-017642-   [Patent document 2] JP 2006-162994-   [Patent document 3] WO2011/108369-   [Patent document 4] WO2011/108370-   [Patent document 5] WO2011/108371-   [Patent document 6] WO2012/050011-   [Patent document 7] WO2012/053355-   [Patent document 8] WO2013/031439

SUMMARY OF INVENTION Technical Problem

In the cases of the above-mentioned laser scan type confocal microscopedevices, typically, a region to be observed is scanned in two dimensionsor three dimensions in the raster scan mode, and the detected lightintensity values are associated with the position information in theregion to be observed so that a microscopic image will be formed. In thecase of this raster scan mode, usually, an image in a region to beobserved is formed by reciprocating a light detection region in acertain one direction within the region to be observed while displacingthe route of the reciprocation of the light detection region in theother direction. In that case, when the detected light intensity isweak, the scanning of the same region is repeated so that the lightintensities will be integrated. In the case of this structure, however,an image of a light-emitting object or a light-emitting particle whichsubstantially remains at rest within a region to be observed during thetime taken by the scanning of the region to be observed can be clearlyformed, but, with respect to a light-emitting object or a light-emittingparticle in a dynamic condition owing to diffusion, transportphenomenon, etc., the position of the light-emitting object orlight-emitting particle is moved during multiple times of the scanningof the region to be observed, and thus, no effective integration effectwill be obtained. On the other hand, in the case of the microscopedevice using evanescent light, since a region to be observed will belimited only to the surface of a cover glass, it is difficult to carryout the imaging of light-emitting objects or light-emitting particles ina thick sample.

By the way, as already noted, according to the way of the scanningmolecule counting method in which, using the optical system of aconfocal microscope or a multiphoton microscope, the existence of alight-emitting particle is detected during moving a light detectionregion in liquid, it is possible to detect faint light from alight-emitting object or a light-emitting particle whose positiondynamically changes in a thick sample. Accordingly, by utilizing the wayof the scanning molecule counting method in the imaging in a microscopicobservation, the formation of an image of a light-emitting object or alight-emitting particle whose position changes dynamically in a thicksample will be achieved.

Thus, the main object of the present invention is to provide amicroscope device, a microscopic observation method and a computerprogram for those, which are capable of the formation of an image of alight-emitting object or a light-emitting particle whose positiondynamically changes in a thick sample by utilizing the way of thescanning molecule counting method.

Solution to Problem

According to one manner of the present invention, the above-mentionedobject is achieved by an optical microscope device which detects lightfrom a light-emitting particle in a sample liquid to detect thelight-emitting particle, using an optical system of a confocalmicroscope or a multiphoton microscope, the device comprising: a lightdetection region mover which moves a position of a light detectionregion multiple times continuously within each observed subregion, therespective observed subregions being obtained by dividing a region to beobserved in a field of view of the microscope into plural regions; alight detector which detects light from the light detection region; anda signal processor which generates time series light intensity data ofthe light from the light detection region detected by the light detectorwhile moving the position of the light detection region in each observedsubregion, detects a signal having a characteristic of a signalindicating light from each light-emitting particle individually in thetime series light intensity data, and determines a position of eachlight-emitting particle corresponding to the detected signal in theregion to be observed.

In the above-mentioned structure, “a light-emitting particle which movesin a sample liquid” may be a particle which emits light, for example, anatom, a molecules or an aggregate of those in a condition that it canmove within an arbitrary liquid, such as in a cell, in a cell organelle.The light-emitting particle is typically a fluorescent particle, but maybe a particle which emits light by phosphorescence, chemoluminescence,bioluminescence, scattering light, etc. The “light detection region” ofthe optical system of a confocal microscope or a multiphoton microscopeis a micro area from which light is detected in those microscopes, andcorresponds to the region to which illumination light is condensed whenit is given from an objective (In a confocal microscope, the “lightdetection region” is defined by the spatial relationship between anobjective and a pinhole). The “region to be observed” is a two- orthree-dimensional region in the field of view of a microscope, in whichregion the imaging is carried out in the present device, and the“observed subregion” is each of a plurality of regions obtained byvirtually dividing the “region to be observed” into plural regions in alattice or grid pattern. Further, the light detector may be a structureusing a photodetector of type which detects the light from a lightdetection region by the photon counting in which the number of photonswhich arrive in every predetermined measuring unit time (bin time) iscounted, and in that case, the time series light intensity data becomestime series photon count data. In this regard, in this specification, a“signal of a light-emitting particle” is meant to be a signal indicatinglight from a light-emitting particle unless noted otherwise.

As understood from the above, in the basic structure of the presentinvention, as in a laser scan type confocal microscope or multiphotonmicroscope, the position of a “light detection region” (confocal volume)is moved within a predetermined two- or three-dimensional space in asample liquid, namely, a two- or three-dimensional space (a “region tobe observed”) in a sample liquid is scanned with the light detectionregion, and thereby, the light emitted from the “region to be observed”is detected. However, in the case of the present invention, especially,the manner of the scanning in the region to be observed with a lightdetection region and the manner of signal processing of the detectedlight differ from in usual cases. Namely, in the present invention, thescanning with the light detection region is performed multiple timescontinuously in the respective observed subregions obtained by virtuallydividing the “region to be observed” in a lattice or grid pattern, andwhen the moving light detection region encompasses a light-emittingparticle in the liquid, the light from the light-emitting particle isdetected (the light measurement of the scanning molecule countingmethod). Then, in the process of the so obtained time series lightintensity data of the detected light, for each of the observedsubregions, the individual detection of a light-emitting particleexisting in the region is performed by the manner of detecting thepresence or absence of a signal of light from a light-emitting particle(Individual detection of a light-emitting particle of the scanningmolecule counting method), and when a light-emitting particle exists,the determination of its position within the region to be observed isconducted. That is, in the structure of the present invention, theprinciple of the scanning molecule counting method is applied for eachobserved subregion so as to perform the detection of the existence of alight-emitting particle in a microscopic observation.

According to this structure, since the size of an observed subregion issufficiently smaller than the size of a region to be observed, the timeto be taken by the scanning of each observed subregion at a scanningspeed which makes it possible to detect weak light from eachlight-emitting particle individually and significantly and determine theposition of each light-emitting particle will become short. Namely, thescanning time taken for the detection of one light-emitting particlebecomes short, and therefore, even in a case that the position of alight-emitting particle changes with time, it can be more surelyachieved to detect significantly the light from the light-emittingparticle multiple times before its position largely changes. Moreover,in the structure of the present invention, since the position of thelight detection region in the sample liquid is not limited to near thesurface of a sample container (unlike the microscopic observation methodwith evanescent light), even for a light-emitting particle moving in aplace away from near the surface of the sample container in a thicksample liquid, it is possible to detect its existence and determine itsposition. What should be understood is that, in the structure of thepresent invention, since the position of a detected light-emittingparticle is determined, the existence position of the light-emittingparticle in a region to be observed can be expressed with a two- orthree-dimensional image. In this regard, with respect to the position ofa light-emitting particle to be determined, first, the position of theobserved subregion in the region to be observed is determinable as theposition of the light-emitting particle. And alternatively, further, ina certain embodiment of detecting a light-emitting particle, theposition of the light-emitting particle in an observed subregion isdeterminable as explained in the column of Embodiments later.

In the above-mentioned structure, in one manner, the multiple times ofsuccessive moving of the position of the light detection region in eachobserved subregion, i.e., the multiple times of scanning with the lightdetection region may be carried out continuously in at least twodirections in each observed subregion. For example, the scanning in theX direction and the scanning in the Y direction may be performedcontinuously in one observed subregion, and also, the scanning in the Xdirection, the scanning in the Y direction and the scanning in the Zdirection may be performed continuously in one observed subregion. Inthat case, as explained in detail in the column of Embodiments later, itbecomes possible to determine the position in the X direction and Ydirection or the position in the X direction, Y direction and Zdirection for a light-emitting particle. Alternatively, the multipletimes of scanning with the light detection region in each observedsubregion may be continuously conducted in the same one direction. Inthat case, if a light-emitting particle exists in an observed subregion,the integration of the light will become possible, or, it becomespossible to acquire information on the moving characteristic ortranslational diffusional characteristic of the light-emitting particlefrom the variation of the position during multiple times of scanning.What is to be understood here is that, since multiple times of scanningis performed in each observed subregion having a small size, thepossibility that one light-emitting particle would deviate from anobserved subregion being scanned in the scanning time becomes low, andthus, the possibility that the light of the same light-emitting particlecan be detected during scanning multiple times becomes higher (Thesuccess rate of detecting the light of the same light-emitting particleduring scanning multiple times becomes higher).

The size of the above-mentioned observed subregion decides the timetaken for the light detection region to scan the observed subregion, andthus, concretely, it may be determined based on the size of the lightdetection region. In this respect, as noted above, in order to catchmore certainly the light of the same light-emitting particle with thelight detection region during scanning multiple times, it is preferableto adjust the size of the observed subregion so that the samelight-emitting particle will not deviate during the scanning time. Thus,preferably, the size of an observed subregion is set such that an(expected) moving length of a light-emitting particle to be detectedwithin a time in which the position of the light detection region ismoved in the observed subregion becomes smaller than the size of theobserved subregion.

Moreover, when the light detection region moves in a certain one waywithin an observed subregion, the scanning time will become shortened ifone time of moving of the light detection region covers the wholeobserved subregion. Thus, it is preferable that the one side length ofan observed subregion is set to be almost the same as the diameter ofthe light detection region. In this respect, as explained in detail inthe column of Embodiments later, in the detection of a light-emittingparticle by the scanning molecule counting method, typically, when alight detection region passes through the existence position of alight-emitting particle, a pulse-like time variation of the lightintensity having a bell-shaped profile is captured as a signal of lightfrom the light-emitting particle. This bell-shaped profile results fromthat the variation of the light intensity, emitted from a light-emittingparticle in the light detection region and detected, depends upon theposition of the light-emitting particle in the light detection region,and the intensity distribution of its light forms a bell-shapeddistribution in which a light intensity reduces as the position of alight-emitting particle moves from the almost center in the lightdetection region toward its periphery. Thus, in order to more certainlycatch a signal of light from a light-emitting particle existing in anobserved subregion, it is preferable that the bell-shaped profile of thesignal of the light from a light-emitting particle appears in timeseries light intensity data generated from the detected light. On theother hand, a light-emitting particle may exist in the position shiftedfrom the center of an observed subregion. Namely, for all light-emittingparticles existing in the whole area of an observed subregion, includingalso a light-emitting particle existing in the position shifted from thecenter of the observed subregion, in order to catch the respectivebell-shaped profiles of those signals, it is necessary to make the edgeportion of the light detection region pass through the whole area of theobserved subregion. For this, it will be preferable that the front edgeand rear edge of a light detection region in its movement direction passthrough the opposite sides (or the opposite faces) of an observedsubregion in the movement direction of the light detection region. Thus,in the above-mentioned present invention, it is more preferable that onetime of moving of the position of a light detection region in eachobserved subregion is performed from when the front edge in the movementdirection of the light detection region passes through one side edge ofthe observed subregion and until the rear edge in the movement directionof the light detection region reaches to the other side edge of theobserved subregion. In this regard, preferably, the manner of this onetime of moving of the position of the light detection region is appliedin all the directions (X, Y, Z) in which the scanning is performed.According to this structure, it becomes possible to determine theposition in an observed subregion of a light-emitting particle whichexists in an observed subregion from the analysis of a bell-shapedprofile of a signal of the light-emitting particle (see the column ofEmbodiments described later).

In the above-mentioned structure, the moving speed of the position ofthe light detection region in its scanning may be appropriatelychangeable based on the characteristics of a light-emitting particle tobe observed or its number density or concentration in the samplesolution. Especially, since the detected light amount will decrease ifthe moving speed of the light detection region becomes quick, it ispreferable that the moving speed of the light detection region can bechanged appropriately so that it becomes possible to measure thedetected light amount with high precision and/or high sensitivity. Inthis connection, more preferably, the moving speed of the position ofthe light detection region is set higher than the diffusion movingvelocity of the light-emitting particle (the average moving speed of theparticle by the Brownian motion). As already noted, in the inventiveobservation technique employing the way of the “scanning moleculecounting method”, the existence of a light-emitting particle is detectedby detecting the light from the light-emitting particle when a lightdetection region encompasses the light-emitting particle, and if thelight-emitting particle moves at random by the Brownian motion, and so,the light-emitting particle would enter into and exit out of the lightdetection region multiple times (during one time of scanning), thesignal expressing the existence of the one light-emitting particle wouldbe detected multiple times, and thereby it would become difficult toassociate a detected signal with the existence of one light-emittingparticle. Thus, as noted above, the moving speed of the light detectionregion is set higher than the diffusion moving velocity of thelight-emitting particle, and thereby, it becomes possible to make onelight-emitting particle correspond with one signal (indicating theexistence of the light-emitting particle). In this regard, since thediffusion moving velocity changes depending upon a characteristic of alight-emitting particle, it is preferable that the moving speed of thelight detection region is changeable appropriately according to thecharacteristic (especially, the diffusing constant) of thelight-emitting particle as noted above.

The moving of the position of the light detection region may be achievedby an arbitrary way. For instance, the position of the light detectionregion may be changed by changing the optical path in the optical systemof the microscope by means of a galvano mirror employed in a laser scantype light microscope, and alternatively, the relative position of thelight detection region in the sample solution may be moved by moving theposition of the sample solution (e.g. by moving the stage of amicroscope). The moving in the direction of the optical axis of anobjective is achievable by adjusting the position of the objective inits height direction or the position of the stage in its heightdirection, or with a mechanism forming the beam light, which enters intoand exits out of the back end of an objective, into a convergent beam ora divergent beam (instead of forming a parallel beam).

By the way, in the above-mentioned inventive microscopic observationtechnique, the way of the individual detection of light-emittingparticles in accordance with the scanning molecule counting method isemployed, and in the scanning molecule counting method, it is possibleto acquire the information on a condition or a characteristic of alight-emitting particle, especially, the size of the light-emittingparticle through making it possible to detect the characteristic of itslight or the occurrence time of the signal of its light by an arbitraryway (patent documents 6-7). Thus, in the above-mentioned presentinvention, the signal processor may be constructed to determine theinformation on the size of a light-emitting particle, by using thecharacteristics of the signals of the same light-emitting particleobtained during multiple times of moving of the light detection regionsin each observed subregion. Such characteristics of the signals of alight-emitting particle, concretely, may be an index value indicating atranslational diffusional characteristic of the light-emitting particleand/or an index value indicating a rotational diffusion characteristicsof the light-emitting particle. In this regard, in this structure, itshould be understood that, in the case of the present invention, theinformation about the size of each light-emitting particle is acquiredunder the condition that the position of the light-emitting particle isdetermined in the region to be observed, or under the condition that theposition of the light-emitting particle is expressed as an image.

Moreover, as noted, for a light-emitting particle detected in thepresent invention, its position in a region to be observed can bedetermined, and can be represented as an image. So, the position of alight-emitting particle can be superimposed and expressed on amicroscopic image (for example, a transmitted light image, anepi-illuminated fluorescence image, etc.) of a region to be observedgenerated by an arbitrary way. Accordingly, in the inventive device,there may be generated a plot image obtained by plotting the position ofa light-emitting particle, whose position in a region to be observed hasbeen determined, on a microscopic image of the region to be observedgenerated by an arbitrary way. According to this structure, it isadvantageous in that the position of a light-emitting particle can beobserved while being superimposed on a microscopic image of a region tobe observed, for example, a microscopic image of a cell or a cellorganelle, acquired by the other way. Also, in that case, when the sizeor the characteristics of a light-emitting particle are detectable, itis expected to more diverse information will be obtained by making itpossible to refer to a characteristic of a light-emitting particle whilebeing superimposed on a microscopic image of a cell or a cell organelle.

Furthermore, according to the “scanning molecule counting method”, it ispossible to count the number of light-emitting particles encompassed inthe light detection region by counting the number of signals (thecounting of particles) and acquire information about a concentration ofthe light-emitting particles. Accordingly, the signal processor in thepresent invention may be designed to determine the number oflight-emitting particles in a region to be observed or the concentrationof light-emitting particles in a liquid based on the number of thedetected light-emitting particles. Also in this structure, it isadvantageous in that the concentration of a light-emitting particle isobservable together with a microscopic image of a region to be observedby another way.

The processes of the microscopic observation technique employing the wayof the scanning molecule counting method in accordance with theabove-mentioned inventive device are realizable with a general computer,also. Accordingly, in accordance with another manner of the presentinvention, there is provided a computer readable storage device having acomputer program product including programmed instructions forobservation with an optical microscope for detecting light from alight-emitting particle in a sample liquid to detect the light-emittingparticle, using an optical system of a confocal microscope or amultiphoton microscope, said programmed instructions causing a computerto perform steps of: moving a position of a light detection regioncontinuously multiple times within each observed subregion obtained bydividing a region to be observed within a field of view of themicroscope into plural regions; detecting light from the light detectionregion by a light detector; and generating time series light intensitydata of the light from the light detection region detected by the lightdetector while moving the position of the light detection region in eachobserved subregion, detecting a signal having a characteristic of asignal indicating light from each light-emitting particle individuallyin the time series light intensity data, and determining a position ofeach light-emitting particle corresponding to the detected signal in theregion to be observed.

Also in this computer program, the moving of the position of the lightdetection region in each observed subregion may be conducted multipletimes continuously in at least two directions and/or continuously in thesame one direction for each observed subregion. The size of the observedsubregion may be determined based on the size of the light detectionregion, and preferably, the size of the observed subregion may be setsuch that the moving length of the light-emitting particle to bedetected within a time in which the position of the light detectionregion is moved in the observed subregion will be smaller than the sizeof the observed subregion. Further, in one embodiment, the length of oneside edge of the observed subregion may be set to be almost equal to thediameter of the light detection region; one time of moving of theposition of the light detection region in each observed subregion may beperformed from when the front edge of the light detection region in itsmoving direction passes through one side edge of the observed subregionand until the rear edge of the light detection region in its movingdirection reaches to the other side edge of the observed subregion. Inthis regard, preferably, the moving speed of the position of the lightdetection region is set higher than the diffusion moving velocity of thelight-emitting particle.

Furthermore, also in the above-mentioned computer program, there may becomprised a procedure of generating a plot image obtained by plottingthe position of the light-emitting particle whose the position in theregion to be observed has been determined in a microscopic image of theregion to be observed generated by an arbitrary way, and/or a procedureof determining information about the size of a light-emitting particleusing a characteristic of a signal(s) of one same light-emittingparticle obtained in the multiple times of moving of the light detectionregion in each observed subregion, where the characteristic of thesignal(s) of the light-emitting particle used for the determination ofthe information about the size of the light-emitting particle may be,for example, an index value indicating a translational diffusionalcharacteristic of the light-emitting particle or an index valueindicating a rotational diffusion characteristic of the light-emittingparticle. Moreover, the above-mentioned computer program may include aprocedure of determining the number of light-emitting particles in theregion to be observed or a concentration of the light-emitting particlein the liquid based on the number of the detected light-emittingparticles.

Furthermore, according to the above-mentioned inventive device orcomputer program, there is realized a novel microscopic observationmethod employing the way of the scanning molecule counting method.Therefore, in accordance with yet other manner of the present invention,there is provided an optical microscopic observation method of detectinglight from a light-emitting particle in a sample liquid to detect thelight-emitting particle, using an optical system of a confocalmicroscope or a multiphoton microscope, comprising steps of: moving aposition of a light detection region continuously multiple times withineach observed subregion obtained by dividing a region to be observedwithin a field of view of the microscope into plural regions; detectinglight from the light detection region by a light detector; andgenerating time series light intensity data of the light from the lightdetection region detected by the light detector while moving theposition of the light detection region in each observed subregion,detecting a signal having a characteristic of a signal indicating lightfrom each light-emitting particle individually in the time series lightintensity data, and determining a position of each light-emittingparticle corresponding to the detected signal in the region to beobserved.

Also in this method, the moving of the position of the light detectionregion in each observed subregion may be conducted multiple timescontinuously in at least two directions and/or continuously in the sameone direction for each observed subregion. The size of the observedsubregion may be determined based on the size of the light detectionregion, and preferably, the size of the observed subregion may be setsuch that the moving length of the light-emitting particle to bedetected within a time in which the position of the light detectionregion is moved in the observed subregion will be smaller than the sizeof the observed subregion. Further, in one embodiment, the length of oneside edge of the observed subregion may be set to be almost equal to thediameter of the light detection region; one time of moving of theposition of the light detection region in each observed subregion may beperformed from when the front edge of the light detection region in itsmoving direction passes through one side edge of the observed subregionand until the rear edge of the light detection region in its movingdirection reaches to the other side edge of the observed subregion. Inthis regard, preferably, the moving speed of the position of the lightdetection region is set higher than the diffusion moving velocity of thelight-emitting particle.

Furthermore, also in the above-mentioned method, there may be compriseda step of generating a plot image obtained by plotting the position ofthe light-emitting particle whose the position in the region to beobserved has been determined in the microscopic image of the region tobe observed generated by an arbitrary way, and/or a step of determininginformation about the size of a light-emitting particle using acharacteristic of a signal(s) of one same light-emitting particleobtained in the multiple times of moving of the light detection regionin each observed subregion, where the characteristic of the signal(s) ofthe light-emitting particle used for the determination of theinformation about the size of the light-emitting particle may be, forexample, an index value indicating a translational diffusionalcharacteristic of the light-emitting particle or an index valueindicating a rotational diffusion characteristic of the light-emittingparticle. Moreover, the above-mentioned method may include a step ofdetermining the number of light-emitting particles in the region to beobserved or a concentration of the light-emitting particle in the liquidbased on the number of the detected light-emitting particles.

Typically, the above-mentioned inventive microscopic observationtechnique is applied to observations, analyses, etc. of conditions ofparticulate, biological objects, e.g., biomolecules, such as protein,peptide, nucleic acid, lipid, sugar chain, amino acid or theseaggregates, viruses, cells, etc., in liquid, such as in a cell or in acell organelle, but the inventive technique may be used for analyses,etc. of conditions of non-biological particles (for example, atoms,molecules, micells, metal colloids, etc.) in an arbitrary liquid, and itshould be understood that such cases belongs to the scope of the presentinvention, also.

Effect of Invention

Thus, according to the present invention, by employing the way of thescanning molecule counting method in the observation and analysis with ascan type optical microscope, it becomes possible to detect and observeindividually a light-emitting particle which exists and moves in theinside of a thick sample liquid with more sufficient accuracy ascompared with the prior art. Especially according to the inventivestructure, sine the position of a detected light-emitting particle canbe determined, it is possible to express the distribution oflight-emitting particles as a two- or three-dimensional image, and thus,it is expected to acquire, from more multidirectional microscopeobservations and analyses thereof, diverse information and knowledgeswhich have not been grasped in the past yet. Especially, in thestructure of the present invention, since the existence distribution oflight-emitting particles can be superimposed and observed on amicroscopic image of a cell or a cell organelle, the present inventionwill be advantageously used for experiments for observing individually abehavior of an arbitrary particle in the inside of a cell or a cellorganelle.

Other purposes and advantages of the present inventions will becomeclear by explanations of the following preferable embodiments of thepresent invention.

BRIEF DESCRIPTIONS OF DRAWINGS

FIG. 1A is a schematic diagram of the internal structure of an opticalmicroscope device employing the scanning molecule counting methodaccording to the present invention. FIG. 1B is a schematic diagram of aconfocal volume (an observation region of a confocal microscope). FIG.1C is a schematic diagram of the mechanism for changing the direction ofthe mirror 7 to move the position of a light detection region in asample (cell). FIG. 1D is a schematic diagram of the mechanism formoving the horizontal position of a micro plate to move the position ofthe light detection region in a sample (cell).

FIGS. 2A and 2B are a schematic diagram explaining about the principleof the light detection in the scanning molecule counting method appliedto the present invention, and a schematic diagram of a time variation ofmeasured light intensity, respectively.

FIG. 3A is a schematic plan diagram of a region to be observed ObR andobserved subregions OsR set in the field of view of a microscope. FIG.3B is a schematic three-dimensional perspective diagram of a region tobe observed and observed subregions. FIG. 3C is a schematic perspectivediagram explaining about the manner of the moving of a light detectionregion which passes through an observed subregion.

FIGS. 4A and 4B are diagrams schematically showing examples of anobserved subregion and a light detection region (upper rows) and timevariations of light intensity (lower rows) when the observed subregionis scanned with the light detection region. FIG. 4C shows a schematicplan diagram illustrating the spatial relationship between an observedsubregion and a light detection region and a drawing illustratingschematically an example of the time variation of light intensity forexplaining about the principle of determining the position (coordinates)of a light-emitting particle in the observed subregion.

FIG. 5 is a drawing showing the detection procedure of a light-emittingparticle in a microscopic observation method performed in accordancewith the present invention in the form of a flow chart.

FIGS. 6A and 6B each are drawings of models in a case that alight-emitting particle crosses a light detection region owing to theBrownian motion and in a case that a light-emitting particle crosses alight detection region by moving the position of the light detectionregion in a sample solution at a velocity quicker than the diffusionalmoving velocity of the light-emitting particle. FIG. 6C shows drawingsexplaining an example of the signal processing step of the detectedsignals in the procedure for detecting the existence of a light-emittingparticle from the measured time series light intensity data (timevariation of photon count) in accordance with the scanning moleculecounting method.

FIG. 7 shows examples of measured photon count data (bar graph); curvesobtained by carrying out the smoothing of the data (dotted line); andGauss functions fitted on the pulse existing region (solid line). In thedrawing, the signals attached with “noise” are disregarded as signalsdue to noises or a contaminant.

FIG. 8 shows examples of simulation results of schematic diagrams ofobserved subregions (8A, 8D); obtained time series light intensity data(8B, 8E); and integration data of light intensity data (8C, 8F) in acase of detecting the position of a light-emitting particle inaccordance with the microscopic observation method in accordance withthe present invention.

FIGS. 8A-8C show a case where a light detection region is moved in the Xdirection, and FIGS. 8D-8F show a case where a light detection region ismoved in the Y direction.

FIG. 9A is a drawing showing schematically an example of a scanningpattern in a raster scan mode in a conventional laser scan type lightmicroscope, and FIG. 9B is a schematic drawing explaining about theobservable area in a microscope using evanescent light.

EXPLANATIONS OF REFERENCE NUMERALS

-   1—Confocal microscope-   2—Light source-   3—Single mode optical fiber-   4—Collimating lens-   5—Dichroic mirror-   6, 7—Galvano mirror-   8—Objective-   9—Micro plate-   10—Well (sample container)-   11—Reflective mirror-   12—Condenser lens-   13—Pinhole-   14—Barrier filter-   14 a—Dichroic mirror-   15—Multi-mode optical fiber-   16, 17—Mirror deflector-   18—Photodetector-   19—Stage position changing apparatus-   20—Computer

DESCRIPTION OF EMBODIMENTS

In the followings, preferable embodiments of the present invention aredescribed in detail.

The Structure of a Microscope Device

In the basic structure, a microscope device which realizes themicroscopic observation technique according to the present invention maybe a device constructed by associating the optical system of a confocalmicroscope with a photodetector, enabling microscope observation withlaser scanning as schematically illustrated in FIG. 1A. Referring tothis drawing, the microscope device 1 consists of an optical system 2-18and a computer 20 for acquiring and analyzing data together withcontrolling the operation of each part in the optical system. Theoptical system of the microscope device 1 may be the same as the opticalsystem of a usual confocal microscope, where laser light, emitted from alight source 2 (Ex), forms a parallel beam with a collimator 2 a, whichbeam is reflected on a dichroic mirror 5 and reflective mirrors (galvanomirror) 6 and 7, entering into an objective 8. Above the objective 8,typically, there is placed a sample container or a micro plate 9 havingwells 10 arranged thereon, to which one to several tens of μL of asample (a solution, cells, etc.) is dispensed, and the laser lightemitted from the objective 8 is focused in the sample liquid in thesample container or well 10, forming a region having strong lightintensity (excitation region). In the sample, when a light-emittingparticle which is an object to be observed, typically, such as afluorescent particle or a particle to which a light-emitting label, suchas a fluorescent dye, is attached, enters into the excitation region,the light-emitting particle is excited and emits light during dwellingin the excitation region. The emitted light (Em) passes through theobjective 8 and the dichroic mirror 5, and is reflected on the mirror 11and condensed by a condenser lens 12, and then the light passes throughthe pinhole 13; transmits through the barrier filter 14 (where a lightcomponent only in a particular wavelength band is selected); and isintroduced into a multimode fiber 15, reaching to the correspondingphotodetector 18, and after the conversion into time series electricsignals, the signals are inputted into the computer 20, where theprocesses of the detection of the signal of a light-emitting particle,the determination of the position of a light-emitting particle and/orother optical analyses are executed in manners explained later. In thisregard, as known in ones skilled in the art, in the above-mentionedstructure, the pinhole 13 is located at a conjugate position of thefocal position of the objective 8, and thereby only the light emittedfrom the focal region of the laser light, i.e., the excitation region,as schematically shown in FIG. 1B, passes through the pinhole 13 whilethe light from regions other than the excitation region is blocked. Thefocal region of the laser light illustrated in FIG. 1B is a lightdetection region, whose effective volume is usually about 1-10 fL inthis microscope device (typically, the light intensity is spread inaccordance with a Gaussian type distribution having the peak at thecenter of the region. The effective volume is a volume of an approximateellipsoid bordering a surface where the light intensity is reduced to1/e² of the center light intensity), which focal region is called as“confocal volume”. Furthermore, in the present invention, since thelight from a single light-emitting particle, for example, the faintlight from one fluorescent dye molecule, is detected, preferably, asuper high sensitive photodetector, usable for the photon counting, isused for the photodetector 18. When the detection of light is performedby the photon counting, the measurement of light intensity is performedfor a predetermined time in a manner of measuring the number of photonswhich have sequentially arrived at a photodetector in every measuringunit time (BIN TIME). Thus, in this case, the time series lightintensity data is time series photon count data. Also, on the stage (notshown) of the microscope, there may be provided a stage positionchanging apparatus 19 for moving the horizontal position of the microplate 9, in order to change the well 10 to be observed. The operation ofthe stage position changing apparatus 19 may be controlled by thecomputer 20. According to this structure, quick measurement can beachieved even when there are two or more specimens.

Furthermore, in the optical system of the above-mentioned microscopedevice, there is further provided a mechanism to scan the inside of thesample liquid with the light detection region, namely to move theposition of the focal region i.e., the light detection region, withinthe sample solution. For this mechanism for moving the position of thelight detection region, for example, there may be employed a galvanomirror device comprising reflective mirrors 6 and 7 and mirrordeflectors 16 and 17 which change the directions of the mirrors 6 and 7,as schematically illustrated in FIG. 1A (the type of moving the absoluteposition of a light detection region). In this case, under control ofthe computer 20, the direction of the reflective mirrors 6 and 7 arechanged as illustrated schematically in FIG. 1C so that the position ofthe light detection region will move, and thereby, for example, itbecomes possible to scan the inside of a cell with the light detectionregion. In this regard, although the galvano mirror device may be thestructurally same as that equipped in a usual laser scan typemicroscope, the manner of the moving of the position of the lightdetection region, i.e. the scanning way is different from the case ofthe usual laser scan type microscope as explained in detail later. Or,alternatively, as illustrated in FIG. 1D, the stage position changingapparatus 19 may be operated in order to move the horizontal position ofthe container 10 (micro plate 9), into which the sample liquid has beendispensed, to move the relative position of the light detection regionin the inside of a cell in the sample (the type of moving the absoluteposition of a sample liquid). The driving of the position of the stageposition changing apparatus 19 is performed by control of the computer20. Further, although not illustrated, a mechanism in which the positionof the light detection region is moved in the vertical direction in thesample liquid may be provided. For such a mechanism, concretely, adevice which moves up and down on the objective 8 or the stage may beemployed. Also, the moving of the position of a light detection regionin the vertical direction is achievable by forming the beam light, whichenters into and exits out of the back end of the objective, into aconverging light or diverging light, instead of a parallel beam. Then,there may be constructed a mechanism that moves the position of thelight detection region in the vertical direction by placing an offsetlens having a variable focal length or a focus variable deformablemirror (not shown) on the optical path of the back end of the objective.

When a light-emitting particle to be an object to be observed emitslight by multiphoton absorption, the above-mentioned optical system isused as a multiphoton microscope. In that case, since the light emissionoccurs only in the focal area of excitation light (light detectionregion), the pinhole 13 may be removed. Moreover, in the microscopedevice 1, there may be provided two or more excitation light sources 2,as shown in the drawing, where the wavelength of excitation light can beappropriately chosen, depending upon the excitation wave length of alight-emitting particle. Similarly, two or more photodetectors 18 may beprovided, where, in a case that two or more kinds of light-emittingparticle having different emission wavelengths are included in thesample, it may be designed that the light from these can be separatelydetected according to the wavelengths. Moreover, although notillustrated, there may be a polarizing beam splitter on the optical pathso that polarized light components of detected light can be separatelydetected for detecting a polarization characteristic a light-emittingparticle as in patent document 7.

The computer 20 has a CPU and a memory, and the inventive procedures areperformed through the CPU executing various operational processings. Inthis regard, each procedure may be done with hardware. All or a part ofprocesses explained in this embodiment may be performed by the computer20 with a computer readable storage device having memorized the programsto realize those processes. Accordingly, the computer 20 may read outthe program memorized in the storage device and realize theabove-mentioned steps by performing the processing and calculations ofinformation. Here, a computer readable storage device may be a magneticdisk, a magnetic optical disk, a CD-ROM, a DVD-ROM, a semiconductormemory, etc. Furthermore, the above-mentioned program may be distributedto a computer through communication line, and the computer which hasreceived this distribution may be made to execute the program.

The Principle of Inventive Microscopic Observation Technique

As described in the column of “Summary of Invention”, in the inventivemicroscopic observation technique, briefly, in the observation with ascan type optical microscope, a region to be observed is divided intoplural observed subregions; each observed subregion is scanned multipletimes one by one in the way of the scanning molecule counting method;and the presence or absence of a light-emitting particle and itsposition are determined. Then, because the position of a light-emittingparticle within a region to be observed is determined, it becomespossible to express the existence distribution of light-emittingparticles as an image (imaging the existence distribution oflight-emitting particles). In the followings, the principles of thescanning molecule counting method and the inventive microscopicobservation technique will be explained.

1. Principle of Scanning Molecule Counting Method

In the scanning molecule counting method (patent documents 3-8),basically, the light detection is performed together with moving theposition of a light detection region CV in a sample, namely, scanningthe inside of the sample solution with the light detection region CV bydriving the mechanism (mirror deflector 17) for moving the position ofthe light detection region to change the optical path or by moving thehorizontal position of the container 10 (micro plate 9) into which thesample liquid is dispensed, as schematically drawn in FIG. 2A. Then, forexample, during the moving of the light detection region CV (in thedrawing, time to-t2), when the light detection region CV passes througha region where one light-emitting particle exists (t1), light is emittedfrom the light-emitting particle, and a pulse form signal havingsignificant light intensity (Em) appears on time series light intensitydata as drawn in FIG. 2B. Thus, by detecting, one by one, each pulseform signal (a significant light intensity variation) appearing asillustrated in FIG. 2B during the execution of the moving of theposition of the light detection region CV and the light detection asdescribed above, the light-emitting particles are detected individually,and by counting the number thereof, the information about the number,concentration or number density of the light-emitting particles existingin the measured region can be acquired. In the principle of thisscanning molecule counting method, no statistical operation processing,like computation of fluctuation of fluorescence intensity, is performed,and light-emitting particles are detected one by one, and therefore,even for a sample in which the concentration of a particle to beobserved is too low to conduct an analysis with sufficient accuracythrough fluorescence correlation spectroscopy (FCS) and fluorescenceintensity distribution analysis (FIDA), the information about theconcentration or the number density of the particles is acquirable.Examples of concrete ways of detecting the signal of a light-emittingparticle from time series light intensity data will be mentioned later.

2. Principle of Inventive Microscopic Observation Technique (i) Overview

As described in the column of “Summary of Invention”, in the case of ausual laser scan type confocal microscope device, the scanning of alight detection region CV in a region to be observed ObR is performed ina raster scan mode, typically, as schematically drawn on FIG. 9A, andthus, the position of the light detection region CV is movedsuccessively over the whole area of the region to be observed ObR. Theposition of the region to be observed, i.e., a scan size, can be set toan arbitrary position in the field of view VF and an arbitrary height inthe direction of the optical axis in the range where the influence ofthe aberration of light is allowable. And, when integration of lightintensity is carried out, for instance, when the detected lightintensity is weak, the light detection region CV is repeatedly movedalong the same scanning route. However, in the case of this structure,if the position of a light-emitting particle detected in the first scanwould have moved in the second or later scan, the significant effect ofthe integration of light intensity could not be obtained. On the otherhand, in the case of a microscope device using evanescent light, insteadof detecting light by the scanning of a micro light detection region CV,it is possible to take an image in a field of view or a region to beobserved into a photodetector, such as a camera, at once, and in thatcase, in the photodetector, such as a camera, the integration of lightintensity or light amount is continuously possible, and therefore, thesignificant effect of the integration of light intensity will beobtained also in a case of the image of a light-emitting particle whoseposition changes. However, in the case of the microscope device usingevanescent light, as schematically drawn in FIG. 9B, the observable areawill be limited to the region near the cover glass surface to which theevanescent light reaches in a sample.

Then, in the present invention, in order to enable detecting alight-emitting particle whose position changes in much broader regions,in a structure of a scan type optical microscope device of the type inwhich the position of a light detection region of a confocal microscopeor a multiphoton microscope moves, a region to be observed ObR within afield of view VF is (virtually) divided into plural observed subregionsOsR as schematically drawn in FIG. 3A or 3B, and in each observedsubregion, for example, in each of regions (X1, Y1), (X2, Y1), - - - ,as shown in FIG. 3B, the scanning with the light detection region isperformed multiple times. In the multiple times of scanning, theposition of the light detection region may be moved in at least twomutually different directions, for example, with reference to FIGS. 3Band 3C, at least one time in each of the X direction S1 and the Ydirection 82, or at least one time in each of the X direction S1, the Ydirection 82 and the Z direction (not shown), and during the scanning,measurement of light intensity from the light detection region may becarried out. Also, in the multiple times of scanning, the position ofthe light detection region may be moved in one direction, for example,at least two times in the X direction S1 or Y direction S2, and duringthe scanning, measurement of light intensity from the light detectionregion may be carried out. Then, in the light intensity data from thelight detection region obtained in time series during the multiple timesof scanning with the light detection region, the detection of the signalof a light-emitting particle is performed in the way of the scanningmolecule counting method explained briefly previously.

In the above-mentioned microscopic observation technique according tothe present invention, the moving range of the position of the lightdetection region can be arbitrarily set in the range where the influenceof the aberration of light is allowable as in the case of a usual laserscan type confocal microscope device, and thus, there are no limitationsin the observable region as in the cases of microscope devices usingevanescent light. Further, the time length taken for scanning with thelight detection region multiple times in each observed subregion islargely shorter as compared with a raster scan mode of a usual laserscan type confocal microscope device, and thus, the detection andlocating of a light-emitting particle moving in a sample, such as alight-emitting particle moving in a cell or a cell organelle can beachieved with more sufficient accuracy. Especially, in a case that theemitted light of a light-emitting particle is weak, and thus, theintegration of light intensity will become effective for detecting itsignificantly, the scanning time of each observed subregion can be setenough short to make it possible to detect the light of the samelight-emitting particle almost at the same position by settingappropriately the relation between the size of an observed subregion anda scanning speed, and thereby, a significant integration effect isexpected. Furthermore, the detection of a light-emitting particle iscarried out in accordance with the principle of the scanning moleculecounting method, where no statistical operation processing like thecomputation of fluorescence intensity fluctuation is performed, andtherefore, the detection of the existence of a particle and theacquisition of information about a concentration or number density ofparticles are achievable even for a sample in which the concentration ofparticles to be observed is too low to conduct analyses with sufficientaccuracy in FCS (Fluorescence Correlation Spectroscopy), FIDA(Fluorescence Intensity Distribution Analysis), etc.

(ii) Setting of the Size of Observed Subregion and the Moving Range ofLight Detection Region

As described in the column of “Summary of Invention”, the time taken fora light detection region to scan an observed subregion is determinedwith the size of an observed subregion, and thus, typically, the size ofan observed subregion is determined based on the size of the lightdetection region. In this respect, if the size of an observed subregionis larger than the size of a light detection region, the whole region ofthe observed subregion cannot be covered by one time of the lightdetection region passaging through the observed subregion, and in thatcase, it would be necessary to make the light detection region passthrough the observed subregion again while including the portion whichhas not been included in the light detection region in the first passagein the observed subregion, and thereby, to do this, the scanning timewould be extended. Therefore, with respect to the size of an observedsubregion, preferably, as schematically drawn in FIG. 3C and FIG. 4A-4C,its one side edge length is set to be equal to the diameter 2W of thelight detection region. In this regard, although the shape of anobserved subregion is formed to be a cube preferably, it should beunderstood that the observed subregion may be a rectangularparallelepiped extending longer in the scanning direction of the lightdetection region (especially, when the scanning is performed only in onedirection).

Moreover, preferably the moving range of the light detection region ineach observed subregion is set such that the light detection regionmoves in one time scanning from the position at which the front edge ofthe light detection region CV in its moving direction Sd touches oneside edge of the observed subregion OsR from its outside to the positionat which the rear edge of the light detection region CV touches theopposite side edge of the observed subregion OsR from its outside asschematically drawn in FIGS. 4A and 4B. In measurement with a confocalmicroscope or a multiphoton microscope, the total amount of the lightfrom the region included in a light detection region is measured as onedata value, where the detected light intensity of a light-emittingparticle changes depending upon the position of the light-emittingparticle in the inside of the light detection region. Namely, usually,the light intensity of a light-emitting particle in the light detectionregion becomes its highest when the light-emitting particle is at thealmost center of the light detection region and decreases as thelight-emitting particle moves toward the edge of the light detectionregion. This spread of the distribution of the light intensity of thelight-emitting particle in the light detection region corresponds to thesize of the light detection region, and follows a point spread function(PSF) determined with the numerical aperture of the objective, theincident beam diameter of excitation light to the objective, thediameter of a pinhole, etc. Accordingly, when a light detection regionpasses through the existence position of a light-emitting particle asalready explained in conjunction with FIG. 2, the light intensity of thelight-emitting particle will change in a bell-shaped pulse form. In thescanning molecule counting method, such a pulse form time variation oflight intensity is detected as a signal of a single light-emittingparticle. Namely, also in the inventive microscopic observationtechnique, as drawn in FIG. 4A, in order to detect the signal of onelight-emitting particle LP with sufficient accuracy, it is preferable tomake the light-emitting particle pass from one edge of the lightdetection region to its other edge so that a bell-shaped pulse form timevariation LS of the light intensity (the width from one skirt edge tothe other skirt edge becomes the diameter 2W of the light detectionregion) will be obtained. On the other hand, as drawn in FIG. 4B, in acase of light-emitting particles α and β existing near the periphery ofthe observed subregion OsR, the bell-shaped pulse form time variations αand β of the light intensity can be detected, respectively, by movingthe light detection region CV from the position at which one edge of thelight detection region CV touches one side edge of the observedsubregion OsR (the left-hand side position) to the position at which theother edge of the light detection region CV touches the opposite sideedge of the observed subregion OsR (the right-hand side position) asshown in the drawings. Therefore, as noted above, the moving range of alight detection region in each observed subregion is preferably therange where the front edge and rear edge of a light detection region inits moving direction touch side edges of an observed subregion from itsoutside, respectively, as in FIG. 4B. That is, the moving length of alight detection region for one observed subregion is set to 4W, i.e.twice of the diameter of the light detection region.

(iii) Determination of the Position of a Light-Emitting Particle

As noted above, when the detection process of a light-emitting particleis performed for each observed subregion, the position of the observedsubregion within a region to be observed is beforehand known at the timeof the setting of the observed subregion, and therefore, the position ofa light-emitting particle detected in each observed subregion will bedetermined at the resolution of the size of the observed subregion.Furthermore, as noted, in a case that the detection of the signal of alight-emitting particle in each observed subregion is carried out by thedetection of a bell-shaped pulse form time variation of the lightintensity (a signal of a light-emitting particle), the position (thetime point) of the peak of the signal of a light-emitting particle isspecified on time series light intensity data. Since a light-emittingparticle is considered to exist on the center axis of the lightdetection region in the direction perpendicular to its moving directionat the position of the peak of the signal of the light-emittingparticle, the position of a light-emitting particle in an observedsubregion can also be determined by detecting the position of the peakof the signal of the light-emitting particle. Concretely, asschematically drawn in FIG. 4C, in time series light intensity dataduring scanning with a light detection region in the X direction, whenthe position of the peak of the signal of a light-emitting particle isdetermined, the coordinate in the X direction of the light-emittingparticle is determined from a gap ΔX of the peak position from thecenter position (which may be the side edge position) of an observedsubregion. Similarly, in time series light intensity data duringscanning with a light detection region in the Y direction, when theposition of the peak of the signal of a light-emitting particle isdetermined, the coordinate in the Y direction of the light-emittingparticle is determined from a gap ΔY of the peak position from thecenter position (which may be the side edge position) of an observedsubregion. Then, the position of the light-emitting particle within aregion to be observed will be determined with the position coordinatesof the observed subregion, and ΔX and ΔY. (Although not illustrated,also in the Z direction, the coordinate in the Z direction of alight-emitting particle may be determined by a gap ΔZ of the peakposition from a specific position of an observed subregion.)

Processing Operation Step of Microscopic Observation

In an embodiment of a microscope observation according to the presentinvention using the microscope device 1 illustrated in FIG. 1A,concretely, there are performed (1) the preparation of a samplecontaining light-emitting particles; (2) a process of measuring lightintensity of the sample and (3) a process of analyzing the measuredlight intensity. FIG. 5 shows processes in this embodiment in the formof flow chart.

(1) Preparation of a Sample

The particle to be an observed object in the inventive microscopicobservation technique may be an arbitrary particle as long as it existsin an arbitrary liquid and it may be a biological molecule, i.e. aprotein, a peptide, a nucleic acid, a lipid, a sugar chain, an aminoacid, etc. or an aggregate thereof, a virus, a cell, a metallic colloidor other non-biological molecules. When the particle to be an observedobject is not a particle which emits no light, there is used a particleobtained by attaching a light emitting label (a fluorescence molecule, aphosphorescence molecule, and a chemiluminescent or bioluminescentmolecule) to the particle to be the observed object in an arbitrarymanner. Typically, the sample is an aqueous solution, but not limited tothis, and it may be an organic solvent or other arbitrary liquids.Further, the particle to be an observed object may be a particle whichexists and moves (changes its position) owing to diffusion or any otherreasons in a cell or a cell organelle. Namely, the sample may be asample liquid used for a usual microscopic observation of a cell or acell organelle.

(2) Measurement of Light Intensity of a Sample Liquid (FIG. 5—Step 100)

In the microscope observation of this embodiment, as noted above,measurement of light intensity is performed together with the moving ofthe position of a light detection region in each observed subregion in aregion to be observed. The moving of the position of the light detectionregion is made by driving the galvano mirror devices 16 and 17 or thestage position changing apparatus 19. In the operation processes,typically, after dispensing a sample into the well(s) 10 of the microplate 9 and putting it on the stage of the microscope, when a userinputs to the computer 20 a command of starting a measurement, thecomputer 20 executes programs memorized in a storage device (not shown)(the process of moving the position of the light detection region in thesample, and the process of detecting light from the light detectionregion during the moving of the position of the light detection region)to start radiating the excitation light and measuring the lightintensity in the light detection region in the sample. During thismeasurement, under the control of the operation process of the computer20 according to the programs, the galvano mirror devices 16, 17 or thestage position changing apparatus 19 drives the galvanomirrors 6, 7 orthe micro plate 9 on the stage of the microscope to move the position ofthe light detection region in the well 10, and simultaneously with this,the photodetector 18 sequentially converts the detected light intoelectric signals and transmits them to the computer 20, which generatesthe time series light intensity data from the transmitted signals andstores them in an arbitrary manner. In this regard, the photodetector 18is typically a super high sensitive photodetector which can detect anarrival of a single photon, and thus when the detection of light isperformed by the photon counting, the time series light intensity datamay be time series photon count data.

As noted, the scanning in the region to be observed with a lightdetection region is performed by each observed subregion. Concretely,for example, the moving of the light detection region may be carried outin each observed subregion (a) at least 1 time in each of the Xdirection and Y direction; (b) at least 1 time in each of the Xdirection, Y direction and Z direction, (c) at least 1 time in each ofthe X direction (or Y direction) and the Z direction, or (d) at least 2times in each of the X direction, Y direction, Z direction. And when thescanning for one observed subregion is completed, the moving of a lightdetection region may be similarly carried out in an adjoining observedsubregion.

The moving speed of the position of the light detection region duringthe light intensity measurement may be a predetermined speed setarbitrarily, e.g. experimentally or so as to meet with an analyticpurpose. The interpretation of a measurement result becomes easier whenthe lapsed time during the measurement and the moving length of theposition of the light detection region are proportional to one another,and thus, basically, it is preferable that the moving speed is constant,but not limited thereto.

By the way, with respect to the moving speed of the position of thelight detection region, in order to perform individual detection oflight-emitting particles from the measured time series light intensitydata or the counting of the number of light-emitting particlesquantitatively with sufficient accuracy, it is preferable to set themoving speed to a value quicker than the moving speed in the randommotion of a light-emitting particle, i.e., the Brownian motion. Since aparticle to be observed in the inventive microscopic observationtechnique is a particle which can move in liquid, its position may movewith time progress according to the Brownian motion. And if the movingspeed of the position of a light detection region is slower than themoving of a particle according to the Brownian motion, as schematicallydrawn in FIG. 6A, the particle moves at random in the region, andthereby, its light intensity changes at random (As already noted, theexcitation light intensity in the light detection region has the peak atthe center of the region and reduces toward the outside), so that itbecomes difficult to specify a significant light intensity variationcorresponding to each light-emitting particle. Thus, preferably, themoving speed of the position of the light detection region is setquicker than the average moving speed of particles by the Brownianmotion (diffusion moving velocity) so that a particle will pass throughthe light detection region in an approximately straight line as drawn inFIG. 6B, and thereby the profile of a light intensity variationcorresponding to each light-emitting particle will form a bell-shapedprofile, as illustrated in the most upper row of FIG. 6C (When alight-emitting particle passes through the light detection region in anapproximately straight line, the profile of the light intensityvariation will be almost the same as the excitation light intensitydistribution), and it can become easier to determine the correspondenceof each light-emitting particle to the light intensity.

Concretely, the time Δt required for a light-emitting particle having adiffusion coefficient D to pass through the light detection region ofradius W (confocal volume) by the Brownian motion is given from theequation of the relation of mean-square displacement:

(2W)²=6D·Δt  (1)

as:

Δt=(2W)²/6D  (2),

and thus, the velocity of the light-emitting particle moving by theBrownian motion (diffusional moving velocity) Vdif, becomesapproximately

Vdif=2W/Δt=3D/W  (3)

Then, with reference to this, the moving speed of the position of thelight detection region may be set to a value sufficiently quicker thanVdif. For example, when the diffusion coefficient of a light-emittingparticle is expected to be about D=2.0×10⁻¹⁰ m²/s, Vdif will be 1.0×10⁻³m/s, supposing W is about 0.62 μm, and therefore, the moving speed ofthe position of the light detection region may be set to its 10 times ormore, 15 mm/s, etc. In this regard, when the diffusion coefficient of aparticle to be observed is unknown, an appropriate moving speed of theposition of the light detection region may be determined by repeatingthe executions of a preliminary experiment with setting various movingspeeds of the position of the light detection region in order to findthe condition that the profile of the light intensity variation becomesan expected profile (typically, similar to the excitation lightintensity distribution).

Furthermore, in a case that the scanning of the light detection regionis performed multiple times in each observed subregion, if alight-emitting particle which exists in an observed subregion in thescanning time is not kept from deviating from the observed subregion, nosignificant integration effect would be obtained, and also, as explainedlater, any analyses using the change of the position of a light-emittingparticle within a scanning time (detection of a diffusing constant,etc.) cannot be performed effectively. Thus, it is preferable that thesize of an observed subregion is set so that the (expected) movinglength of a light-emitting particle to be detected within a time inwhich the position of the light detection region is moved in an observedsubregion will be smaller than the size of an observed subregion.However, as noted above, preferably, one side edge length of an observedsubregion is to be set to almost equal to the diameter of the lightdetection region. Then, in practical, the scanning speed may be set togive the scanning time in which the moving length of a light-emittingparticle by its diffusion will not exceed beyond the size of theobserved subregion. Concretely, the scanning time ΔT of a lightdetection region in k times of scanning a route, whose length in onetime scanning is 4W, at the moving speed v is given by:

ΔT=4W−k/v  (4)

On the other hand, the moving length x of the light-emitting particle bydiffusion in the scanning time ΔT is given, similarly to Exp. (1) by:

<x> ²=2DΔT  (5)

Here, the conditions of x²≦W² should be established, and thus, it ispreferable that the scanning speed is set so that

v≦8D−k/W  (6)

will be established.

(3) Analysis Processing of Light Intensity

When the time series light intensity data is generated through thescanning of each observed subregion, there may be carried out thedetection of a signal of a light-emitting particle, the counting oflight-emitting particles, the determination of the position of alight-emitting particle, and other various analyses (concentrationcalculation etc.), using the light intensity values of the time serieslight intensity data, as described below

(i) Individual Detection of Signal of Light-Emitting Particles (FIG.5—Steps 110-160)

As already noted, when the track of one light-emitting particle in itspassing through the light detection region is approximately straight asshown in FIG. 6B, the light intensity variation in the signalcorresponding to the particle on the time series light intensity datahas a bell shaped profile reflecting the light intensity distribution inthe light detection region determined by the optical system. Thus,basically in the scanning molecule counting method, when the time widthΔτ for which the light intensity value exceeding an appropriately setthreshold value Ith continues is in a predetermined range on the lightintensity data, the signal having the profile of the light intensity maybe judged to correspond to one particle having passed through the lightdetection region, and thereby one light-emitting particle is detected.And a signal whose light intensity does not exceed the threshold valueIth or which does not have time width Δτ in the predetermined range isjudged as noise or a signal of a contaminant. Further, when the lightintensity distribution in the light detection region can be assumed asGaussian distribution:

I=A·exp(−2t ² /a ²)  (7),

and when the intensity A and the width a, computed by fitting Expression(7) to the profile of a significant light intensity (a profile which canbe clearly judged not to be a background), are within the respectivepredetermined ranges, the profile of the light intensity may be judgedto correspond to one particle having passed through the light detectionregion, and thereby the detection of one light-emitting particle will bedone (The signal with the intensity A and the width a out of thepredetermined ranges may be judged as a noise or a contaminant signaland ignored in the later analysis, etc.).

As an example of the process of the detection of signals on lightintensity data, first, a smoothing treatment is performed to the timeseries light signal data (FIG. 6C, the most upper row “detected result(unprocessed)”) (FIG. 5—step 110, FIG. 6C mid-upper row “smoothing”).Although the light emitted by a light-emitting particle is stochastic sothat gaps will be generated in data values in minute time, such gaps inthe data value can be disregarded by the smoothing treatment. Thesmoothing treatment may be done until the gaps in the data value asmentioned can be disregarded, for example, by the moving average method.In this regard, parameters in performing the smoothing treatment, e.g.,the number of datum points in one time of the averaging, the number oftimes of a moving average, etc. in the moving averages method, may beappropriately set in accordance with the moving speed (scanning speed)of the position of the light detection region and/or BIN TIME in thelight intensity data acquisition.

Next, on the light intensity data after the smoothing treatment, inorder to detect a time domain (pulse existing region) in which asignificant pulse form signal (referred to as “pulse signal” hereafter)exists, the first differentiation value with time of the light intensitydata after the smoothing treatment is computed (step 120). Asillustrated in FIG. 6C, the mid-low row “time differential”, in the timedifferential value of light signal data, the variation of the valueincreases at the time of the signal value change, and thereby, the startpoint and the end point of a significant signal can be determinedadvantageously by referring to the time differential value.

After that, a significant pulse signal is detected sequentially on thelight intensity data (Steps 130-160). Concretely, first, on thetime-differential value data of the light intensity data, the startpoint and the end point of one pulse signal are searched and determinedby referring to the time differential value sequentially, so that apulse existing region will be specified (step 130). When one pulseexisting region has been specified, the fitting of a bell-shapedfunction is applied to the smoothed light intensity data in the pulseexisting region (FIG. 6C, the lower row “bell-shaped function fitting”),and then, parameters of the pulse of the bell-shaped function, such asthe peak intensity (the maximum), Ipeak; the pulse width (full width athalf maximum), Wpeak; the correlation coefficient in the fitting (of theleast square method), etc. are computed (step 140). In this regard,although the bell-shaped function to be used in the fitting is typicallyGauss function, it may be Lorentz type function. And, it is judgedwhether or not computed parameters of the bell shaped function are inthe corresponding predetermined ranges assumed for the parameters of abell shaped profile drawn by a pulse signal detected when onelight-emitting particle passes through the light detection region,namely, whether or not the peak intensity, pulse width and correlationcoefficient are in the corresponding predetermined ranges, respectively(step 150). Accordingly, as shown in FIG. 7 left, the signal, whosecomputed parameters of the bell-shaped function are judged to be withinthe ranges assumed in a signal corresponding to one light-emittingparticle, is judged as a signal corresponding to one light-emittingparticle, and thereby one light-emitting particle is detected. On theother hand, a pulse signal, whose computed parameters of the bell-shapedfunction are not within the assumed ranges, as shown in FIG. 7 right, isdisregarded as noise. In this regard, simultaneously with detection ofsignal(s) of (a) light-emitting particle(s), the counting of the numberof signals, i.e., the counting of light-emitting particles, may beperformed.

The searching and the judgment of a pulse signal in the above-mentionedprocesses of steps 130-150 may be repetitively carried out in the wholeregions of the light intensity data of each observed subregion (step160). In a case that mutually different components of the light aredetected separately and two or more time series light intensity data aregenerated, the processes of steps 130-150 may be performed for each timeseries light intensity data. In this regard, the process of detecting asignal of a light-emitting particle from light intensity dataindividually may be performed by an arbitrary way, other than theabove-mentioned processes.

(ii) Integration of Signals of the Same Light-Emitting Particle (FIG.5—Step 170)

By the way, in the microscopic observation technique of this embodiment,when the scanning in the same direction is performed multiple times foreach observed subregion, it is expected that signals of the samelight-emitting particle have appeared repeatedly on time series lightdetection data. In that case, the improvement in the accuracy of thesignal of a light-emitting particle is expected by integrating the datavalues on time series light detection data or the data values of theregions detected as a signal of a light-emitting particle along thescanning route while the positions on the scanning route in the datavalues will be in agreement with one another. In one of concrete mannersof such a integration, as noted, after detecting individually signals oflight-emitting particles on time series light detection data in eachobserved subregion, the light intensity values of the signals of adetected light-emitting particle are integrated, and their total valueor average value may be used as the light intensity value of the signalof the light-emitting particle. The processes for extracting the signalsof the same light-emitting particle in the case of the integration oflight intensity values may be performed, for example, in the waydescribed in patent documents 6-7.

For an alternative manner of the integration of time series lightdetection data, the integration of data values along the scanning routemay be carried out prior to the individual detection of signals oflight-emitting particles on the time series light detection data (step105). In this case, especially when the light intensity value of alight-emitting particle obtained in one time scanning is weak, the lightintensity value for the light-emitting particle increases by integrationof the data values, and thereby, improvement in the accuracy of theindividual detection of the signal of a light-emitting particle isexpected. FIG. 8 is diagrams showing the simulation examples ofprocesses in the cases of performing individual detection of a signal ofa light-emitting particle after the integration of data values along ascanning route. In this regard, FIGS. 8A and 8D each are drawingsschematically showing the motion of a light-emitting particle LP with adiffusing constant D=1 μm²/sec in a condition that the light-emittingparticle moves by the Brownian motion within the observed subregion OsRof 0.4 μm square while a light detection region CV repetitively moves at6.4 mm/sec in speed (0.8 μm in amplitude, 4 kHz in frequency) in the Xdirection (S1+, S1− in FIG. 8A) and in the Y direction (S2+, S2− in FIG.8D); and FIG. 8B, the upper row, and FIG. 8E, the upper row, areexamples of photon counts PC obtained in the scanning. In the cases ofthese examples, the scanning time in scanning five times will be 1.25msec, and thus, the displacement by diffusion becomes about 0.09 μm.

With reference to FIG. 8, as understood from FIG. 8B upper row and FIG.8E upper row, as noted above, when photon detection is performed duringscanning, the signal of the same light-emitting particle LP appearrepeatedly in time series photon count data PC (light intensity data).However, in the illustrated examples, the moving direction of the lightdetection region turns to be reverse in scanning in each even-numberedtime. Thus, before the integration of data, reversing the time axis isperformed for the data area corresponding to the periods in which themoving direction of the light detection region was reversed as shown inFIG. 8B lower row, and FIG. 8E lower row. After this, in the time seriesphoton count data of FIG. 8B lower row, and FIG. 8E lower row, theintegration of data values is performed under the condition that theposition on the scanning route is to be matched, namely, the regionscorresponding to the respective arrows in FIG. 8B lower row, and FIG. 8Elower row will be overlapped mutually. Then, as illustrated in FIGS. 8Cand 8F, since the signal intensity of a light-emitting particle LPincreases, an improvement in the accuracy of individual detection of thesignal of a light-emitting particle (the accuracy in discriminatingbetween a signal and a noise) will be expected. In this regard, in timeseries photon count data of each observed subregion, the concreteprocess for improving the accuracy of the signal detection of alight-emitting particle may be performed in the way described in patentdocument 8.

(iii) Determination of Position of Light-Emitting Particle (FIG. 5—Step180)

As already noted, when the signal of a light-emitting particle isdetected in each observed subregion, since the position (coordinates) ofeach observed subregion OsR in the region to be observed ObR is known,the position of the light-emitting particle is determined at theresolution of the size of the observed subregion. Furthermore, from thedistance between the appearance position of the peak of the signal ofthe light-emitting particle and a specific position (the center or anedge) of an observed subregion, the position (coordinates) of alight-emitting particle can be determined in detail (refer to FIG. 4C).Thus, when the position of a light-emitting particle is determined, byusing this information, it will become possible to express the existencedistribution of light-emitting particles in the region to be observedObR as an image. Moreover, the existence distribution image oflight-emitting particle may be represented while being superimposed on amicroscopic image of the region to be observed ObR obtained by the otherarbitrary microscopic observation method (phase-contrast microscopy,differential interference microscopy, epifluorescent microscopy, etc.)on the display of the computer 20. Concretely, there may be generated aplot image obtained by plotting the positions of light-emittingparticles in a microscopic image obtained by an arbitrary microscopicobservation method.

(iv) Determination of Light-Emitting Particle Concentration

When the number of light-emitting particles is determined by countingthe number of signals of detected light-emitting particles, if the wholevolume of the region through which the light detection region has passedis further computed by an arbitrary way, the concentration of thelight-emitting particle can be determined from the volume value and thenumber of light-emitting particles. The whole volume of the regionthrough which the light detection region has passed may be determined,for example, in the way described in patent document 1.

(v) Estimation of Size of Light-Emitting Particle

In the scanning molecule counting method, as described in patentdocument 6, when the signals of the same light-emitting particle havebeen detected multiple times, the displacement of the light-emittingparticle in the scanning period is detected using the time points of thegenerations of those signals (at the times of the peaks), and thetranslational diffusional characteristic (for example, a diffusingconstant) of the light-emitting particle can be estimated from thedisplacement. The translational diffusional characteristic is a functionof the size of a light-emitting particle, and thereby, the estimation ofthe size of the detected light-emitting particle becomes possible. Thus,also in the inventive microscopic observation technique, usinggeneration time points of signals of a light-emitting particle detectedwithin each observed subregion, the estimation of a translationaldiffusional characteristic of the light-emitting particle and the sizeof the light-emitting particle may be performed. Its concrete processesmay be performed in the way described in patent document 6. Moreover, asin the example of FIG. 8, in a case that the integration of lightintensity is carried out before the detection of a signal of alight-emitting particle, it is possible to estimate a translationaldiffusional characteristic with reference to the width d of theintegrated signal of the light-emitting particle (The larger thediffusing constant of a light-emitting particle is, the larger itsdisplacement within scan time becomes, and the width d of the integratedsignal increases).

In addition, in the scanning molecule counting method, the polarizationcharacteristic or a rotational diffusion characteristic (for example,polarization degree) of a light-emitting particle can be measured bydetecting polarized light components of detected light separately asdescribed in patent document 7. Especially, because a rotationaldiffusion characteristic is a function of the size of a light-emittingparticle, the size of a detected light-emitting particle can beestimated. Then, also in the inventive microscopic observationtechnique, the estimation of the polarization characteristic or therotational diffusion characteristic of a light-emitting particle and thesize of the light-emitting particle, detected within each observedsubregion, by detecting the light from a light detection region whiledividing it into two or more polarized light components and using thepolarized light component intensities. Concrete processed may beperformed in the way described in patent document 7.

As noted above, when the concentration, translational diffusionalcharacteristic, polarization characteristic or rotational diffusioncharacteristic and/or size of a light-emitting particle are estimated,those values are represented in conjunction with an image expressing theexistence distribution of the light-emitting particle. Alternatively,the respective characteristic values obtained in each observed subregionmay be displayed as an image expressing the distribution of theconcentration, translational diffusional characteristic, polarizationcharacteristic or rotational diffusion characteristic and/or size of thelight-emitting particle. Moreover, these characteristics of thelight-emitting particle may be represented while being superimposed on amicroscopic image of a cell or a cell organelle, etc., obtained by theother arbitrary microscopic observation method. Thereby, it becomespossible to grasp a characteristic of a light-emitting particle whichexists in a cell or a cell organelle while its position in the cell orthe cell organelle is specified.

Thus, according to the above-mentioned inventive microscopic observationtechnique, by conducting multiple times of scanning with a lightdetection region in each observed subregion in a region to be observed,and carrying out the individual detection of light-emitting particles bythe scanning molecule counting method, the formation of an imageexpressing the existence distribution of light-emitting particles whosepositions vary dynamically in a thick sample becomes possible. Accordingto this feature, especially, it becomes possible to detect individuallythe existence position of a light-emitting particle in a cell or a cellorganelle, and this is expected to be used advantageously in theresearch of cells and/or cell organelles, etc.

1. An optical microscope device which detects light from alight-emitting particle in a sample liquid to detect the light-emittingparticle, using an optical system of a confocal microscope or amultiphoton microscope, the device comprising: a light detection regionmover which moves a position of a light detection region multiple timescontinuously within each observed subregion, the respective observedsubregions being obtained by dividing a region to be observed in a fieldof view of the microscope into plural regions; a light detector whichdetects the light from the light detection region; and a signalprocessor which generates time series light intensity data of the lightfrom the light detection region detected by the light detector whilemoving the position of the light detection region in each observedsubregion, detects a signal having a profile of a light intensityvariation indicating light from each light-emitting particleindividually in the time series light intensity data, and determines aposition of each light-emitting particle corresponding to the detectedsignal in the region to be observed.
 2. The device of claim 1, whereinthe moving of the position of the light detection region in eachobserved subregion is conducted continuously in at least two directionsfor each observed subregion.
 3. The device of claim 1, wherein themoving of the position of the light detection region in each observedsubregion is conducted multiple times in one same direction for eachobserved subregion.
 4. The device of claim 1, wherein the size of theobserved subregion is determined based on the size of the lightdetection region.
 5. The device of claim 1, wherein a size of theobserved subregion is set such that a moving length of a light-emittingparticle to be detected within a time in which the position of the lightdetection region is moved in the observed subregion becomes smaller thanthe size of the observed subregion.
 6. The device of claim 1, wherein alength of one side edge of the observed subregion is almost equal to adiameter of the light detection region; and one time of the moving ofthe position of the light detection region in each observed subregionsis carried out from when a front edge in a moving direction of the lightdetection region passes through one side edge of the observed subregionand until a rear edge in the moving direction of the light detectionregion arrives at another side edge of the observed subregion.
 7. Thedevice of claim 1, wherein the device produces a plot image obtained byplotting a position of the light-emitting particle whose position in theregion to be observed has been determined in a microscopic image of theregion to be observed generated by an arbitrary way.
 8. The device ofclaim 1, wherein the signal processor determines information about asize of the light-emitting particle by using a characteristic of signalsof one same light-emitting particle obtained through the multiple timesof moving of the light detection region in each observed subregion. 9.The device of claim 8, wherein the characteristic of the signals of thelight-emitting particle used for determining the information about thesize of the light-emitting particle is an index value expressing atranslational diffusional characteristic of the light-emitting particleor an index value expressing a rotational diffusion characteristic ofthe light-emitting particle.
 10. The device of claim 1, wherein thesignal processor determines the number of the light-emitting particlesin the region to be observed or a concentration of the light-emittingparticle in the liquid based on the number of the detectedlight-emitting particles.
 11. An optical microscopic observation methodof detecting light from a light-emitting particle in a sample liquid todetect the light-emitting particle, using an optical system of aconfocal microscope or a multiphoton microscope, comprising: (a) movinga position of a light detection region continuously multiple timeswithin each observed subregion obtained by dividing a region to beobserved within a field of view of the microscope into plural regions;(b) detecting light from the light detection region by a light detector;and (c) generating time series light intensity data of the light fromthe light detection region detected by the light detector while movingthe position of the light detection region in each observed subregion,detecting a signal having a profile of a light intensity variationindicating light from each light-emitting particle individually in thetime series light intensity data, and determining a position of eachlight-emitting particle corresponding to the detected signal in theregion to be observed.
 12. A computer readable storage device having acomputer program product including programmed instructions forobservation with an optical microscope for detecting light from alight-emitting particle in a sample liquid to detect the light-emittingparticle, using an optical system of a confocal microscope or amultiphoton microscope, said programmed instructions causing a computerto perform steps of: moving a position of a light detection regioncontinuously multiple times within each observed subregion obtained bydividing a region to be observed within a field of view of themicroscope into plural regions; detecting light from the light detectionregion by a light detector; and generating time series light intensitydata of the light from the light detection region detected by the lightdetector while moving the position of the light detection region in eachobserved subregion, detecting a signal having a profile of a lightintensity variation indicating light from each light-emitting particleindividually in the time series light intensity data, and determining aposition of each light-emitting particle corresponding to the detectedsignal in the region to be observed.